Solarus 950 plasma cleaner

Translation initiation complex was programmed using 2 μM P. aeruginosa 70 S ribosome, 25 μM mRNA, 10 μM fMet-tRNA_i^fMet and incubated in final 70 S buffer (10 mM Hepes 7.6, 60 mM NH₄Cl, 15 mM KCl, 10 mM MgCl₂, 1 mM β-mercaptoethanol) at 37 °C for 15 min. The ternary complex was then incubated with 30 μM IF2 and 2 mM GDPCP (Millipore Sigma) at room temperature for 15 min. The mixture containing 2 μM P. aeruginosa 70S-IC (4 μl) was applied to Quantifoil R2/1 gold 200 mesh grids (Electron Microscopy Sciences) which were pre-cleaned in a Solarus 950 plasma cleaner (Gatan). The grids were plunged-frozen in liquid nitrogen-cooled ethane using a Leica EM GP2 cryo-plunger. Grids were transferred into a Titan Krios G3i electron microscope (ThermoFisher Scientific) operating at 300 keV and equipped with a GIF Quantum LS energy filter (Gatan) and a K3 direct electron detector camera (Gatan). The image stacks (movies) were acquired in super-resolution mode with pixel size of 0.425 Å/pixel. Data collection was done in the EPU software (ThermoFisher Scientific) setup to record movies with 31 fractions with a total accumulated dose of ~31 e^-/Ų/movie. A total of 8056 image stacks were collected with a defocus ranging between –0.2 and –2.1 µm. The statistics of data acquisition are summarized in Supplementary Table 1.

Translation initiation complex was programmed using 2 μM P. aeruginosa 70 S ribosome, 25 μM mRNA, 10 μM fMet-tRNA_i^fMet and incubated in final 70 S buffer (10 mM Hepes 7.6, 60 mM NH₄Cl, 15 mM KCl, 10 mM MgCl₂, 1 mM β-mercaptoethanol) at 37 °C for 15 min. The ternary complex was then incubated with 30 μM IF2 and 2 mM GDPCP (Millipore Sigma) at room temperature for 15 min. The mixture containing 2 μM P. aeruginosa 70S-IC (4 μl) was applied to Quantifoil R2/1 gold 200 mesh grids (Electron Microscopy Sciences) which were pre-cleaned in a Solarus 950 plasma cleaner (Gatan). The grids were plunged-frozen in liquid nitrogen-cooled ethane using a Leica EM GP2 cryo-plunger. Grids were transferred into a Titan Krios G3i electron microscope (ThermoFisher Scientific) operating at 300 keV and equipped with a GIF Quantum LS energy filter (Gatan) and a K3 direct electron detector camera (Gatan). The image stacks (movies) were acquired in super-resolution mode with pixel size of 0.425 Å/pixel. Data collection was done in the EPU software (ThermoFisher Scientific) setup to record movies with 31 fractions with a total accumulated dose of ~31 e^-/Ų/movie. A total of 8056 image stacks were collected with a defocus ranging between –0.2 and –2.1 µm. The statistics of data acquisition are summarized in Supplementary Table 1.

The sample was exchanged to the storage buffer without glycerol for cryoEM studies. The initial sample quality was assessed via negative staining. For negative staining, the continuous carbon copper grids (Electron Microscopy Science) were glow discharged in a Gatan Solarus 950 Plasma cleaner for 20 seconds. 4 μL of the sample was applied to the glow discharged grid and stained with 2% (w/v) uranyl acetate. The ice-embedded specimens were prepared by placing 3 μL of 0.5 mg/ml each complex onto a glow-discharged Quantifoil grids (R 2/2 Jena, Germany). The grids were blotted for 3 seconds at 4 °C in 100% humidity before being plunged into liquid ethane by the use of an FEI Vitrobot MK IV (Hillsboro OR).

UltrAuFoil R 1.2/1.3 grids (Au, 300 mesh; Quantifoil Micro Tools GmbH) were used for sample vitrification. The grids were treated with Ar/O₂ plasma (Solarus 950 plasma cleaner, Gatan) for 10 s immediately before sample application. A total of 0.5 μl of 0.04 mM lauryl maltose neopentyl glycol stock solution was mixed with 3.5 μl of the complex, and 3 μl was immediately loaded onto the grid. Grids were prepared using Vitrobot mark IV (Thermo Fisher Scientific). Temperature inside the chamber was maintained at 10°C, while humidity was at 100%. Blotting force was set to 0, wait time to 10 s, while the blotting time was varied within a 4- to 7-s range. Following the blotting step, the grids were plunge frozen into liquid ethane and cooled by liquid nitrogen.

Postfusion hMPV F was heat-treated at 70 °C for 10 min, then applied to a CF-400-Cu grid (Electron Microscopy Sciences) that had been plasma cleaned for 45 s in a Solarus 950 plasma cleaner (Gatan) with a 4:1 ratio of O₂/H₂. The grid was stained using methylamine tungstate (Nanoprobes). Prefusion-stabilized hMPV F DS-CavEs2 was incubated with a two-fold molar excess of MPE8 Fab in 1× PBS at room temperature for 30 min. The hMPV-F:Fab complexes were diluted to a concentration of 0.03 mg/mL in 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN₃, then deposited on a CF-400-Cu grid. Grids were imaged at a magnification of 92,000× (corresponding to a calibrated pixel size of 1.63 Å/pix) in a Talos F200C TEM microscope equipped with a Ceta 16 M detector (Thermo Fisher Scientific). CTF-estimation and particle picking were performed in cisTEM³⁵ . Particles were then exported to cryoSPARC v2.15.0 for 2D classification^{36 (link).}